Project Summary Mycobacterium tuberculosis (MTB) is responsible for more deaths than any other infectious disease in the world. Despite decades of investigation, little is known about the early interactions of MTB and the innate immune system. Previous investigations have relied on bulk tissue methods that are limited in resolution within a physiologic in vivo model and require further investigations ex vivo. Our group has investigated the cellular recruitment of innate immune cells using these methods. We have shown mononuclear phagocytes (MPs) recruited during MTB infection are heterogeneous and dynamic through time. In early infection, preliminary results demonstrate a differential cell type involvement and rate of MTB cell-to-cell transfer depending on the bacterial strain. However, our results are limited by the ability to investigate mechanisms and resolution to describe unique cellular subtypes. Single-cell RNA sequencing (scRNA-seq) is an increasingly utilized method that provides detailed description of cell types and cellular alterations in disease models. In this application, we propose utilizing scRNA-seq for the first time in MTB-infected mice. This technology will allow us to describe the mononuclear phagocyte population involved in the early stages of MTB infection, and elucidate which cell types are conducive or resistant to MTB growth. We will investigate changes in this population in the absence of T cells and in the absence of a critical MTB virulence factor, ESX-1. In addition, we will describe the cellular pathway of infection over time to elucidate cell-to-cell transfer in the presence and absence of T cells and ESX- 1. In this way, we can begin to understand both the role of the host and the bacteria in the early stages of infection, and provide essential information for the development of an effective vaccine against infection via intercepting MTB cellular transfer.